In humans and other mammals, 1-10% of fertilized eggs become triploid (and die) most often due to fertilization by more than one sperm. This is normally prevented by the release of egg cortical granules (CGs) whose contents modify the zona pellucida (ZP). The Aims will determine the biochemical basis for the failure of CG release in pre-ovulatory oocytes, and the roles of intracellular calcium (Ca2+) oscillations in CG release and cell cycle resumption in mature mouse eggs, as follows. 1) In mature eggs, the hypothesis will be tested that CG exocytosis and cell cycle resumption have different Ca2+ requirements encoded in Ca2+ oscillation parameters. Although Ca2+ oscillations are known to cause CG release, ZP modifications, and cell cycle resumption (via decreases in H1 and MAP kinases) upon fertilization, it is not known whether these events require different numbers or types of oscillations for their activation and/or timing. Three hypotheses will be tested by experimentally changing the amplitude, frequency, or number of oscillations, and quantifying changes in the aforementioned events of egg activation. 2) In pre ovulatory oocytes, the blocked step in the CG secretory pathways will be identified. Three hypothesis will be investigated: (A) Failure of CG translocation from the cortex to the plasma membrane, (B) normal translocation followed by a failure in CG docking, and (C) failure of membrane fusion after translocation/docking. For the blocked step (A, B, or C), relevant structural and regulatory proteins will be compared in secretion-competent mature eggs and incompetent oocytes for protein amount, localization, and phosphorylation status to begin to identify the biochemical deficiency responsible for the failure of exocytosis (including secretory-machinery proteins, calmodulin-depth kinase II, synapsin, & relevant cytoskeletal proteins). Significance (of long-range objectives): Aim 1: The precise Ca2+ requirements (this study) coupled with the identification of the Ca2+ effectors (Aim 2 & other studies) will provide fundamental information about the molecular mechanism by which Ca2+ causes egg activation events responsible for the onset of mammalian development. Using the methods in the proposal, in vitro matured human oocytes could be tested for activation competence without sperm (not applied for herein). Aim 2: Identification of the proteins involved in secretion and the (pre-ovulatory) biochemical change(s) responsible for CG release will provide valuable markers of oocyte maturation to evaluate emerging protocols to mature animal and human oocytes in vitro or in vivo.